Project Summary/Abstract High blood pressure is often associated with excessive salt and fluid retention from the kidney. The peptide hormone angiotensin II (Ang II) is one of the most important factors in maintaining body salt and fluid and blood pressure homeostasis by regulating salt and fluid reabsorption from proximal tubules of the kidney. Thus increased production and actions of Ang II in proximal tubules can cause salt and fluid retention and consequently increase blood pressure. Ang II exerts powerful effects on proximal tubular sodium and fluid transport by activating cell surface receptors on apical and basolateral membranes of proximal tubule cells. However, we have evidence that a) circulating and paracrine Ang II is taken up by proximal tubule cells in culture or by the kidney via an AT1 (AT1a) receptor-mediated mechanism;b) microinjection of Ang II directly into the cells can induce intracellular calcium responses;and c) intracellular Ang II can induce mRNA expression of the major sodium and hydrogen antiporter, NHE-3, in isolated rat renal cortical nuclei. In this project, we hypothesize that circulating and paracrine Ang II is taken up by proximal tubule cells through AT1a receptor-mediated endocytosis, and that after endocytosis, Ang II acts as an intracellular hormone to increase the expression and activity of NHE-3, increase salt and fluid reabsorption by proximal tubules, and thereby induce hypertension. This hypothesis will be tested in four specific aims. Is Specific Aim I, we will test the hypothesis that in polarized proximal tubule cells, apical membrane AT1a receptors play a dominant role in mediating intracellular uptake of luminal Ang II, whereas basolateral AT1a receptors play a relatively minor role in mediating intracellular uptake of interstitial Ang II in vitro. In the absence of apical AT1a receptors, AT1b receptors or the endocytic receptor megalin are unable to assume the role of AT1a receptors. In Specific Aim II, we will test the hypothesis that in polarized proximal tubule cells, apical membrane AT1a receptor-mediated intracellular uptake of Ang II is primarily regulated by the non-canonical cytoskeleton microtubule (microtubule-associated proteins, MAPs)- and caveolin-1-dependent mechanisms, rather than by the canonical clathrin-coated pits-dependent pathway. In Specific Aim III, we will test the hypothesis that in polarized proximal tubule cells, expression of an intracellular Ang II fusion protein induces the expression and increases the activities of NHE-3 in AP membranes via the activation of PLC/Ca2+/PKC[unreadable]/[unreadable]2 and calcineurin/NF-[unreadable]B signaling pathways. Finally, Specific Aim IV will test the hypothesis that intrarenal adenoviral gene transfer of an intracellular Ang II fusion protein selectively in proximal tubules increases salt and fluid reabsorption, promotes salt and fluid retention, and thereby increases blood pressure by increasing the expression and activity of apical NHE-3 via activation of AT1 (AT1a) receptors. These studies will provide new insights into important roles of intracellular or intracrine Ang II in the regulation of salt and fluid transport in proximal tubules of the kidney and in the pathogenesis of hypertension.